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E H Harrison

Publications and source records attributed to E H Harrison.

At least 37 records · Page 2Linked to original sources
PubMed1997

Lecithin:retinol acyltransferase and retinyl ester hydrolase activities are differentially regulated by retinoids and have distinct distributions between hepatocyte and nonparenchymal cell fractions of rat liver.

The cellular distribution of enzymes that esterify retinol and hydrolyze retinyl esters (RE) was studied in liver of vitamin A-sufficient, -deficient, and deficient rats treated with retinoic acid or N-(4-hydroxyphenyl)-retinamide. Livers were perfused and cell fractions enriched in hepatocytes, and nonparenchymal cells were obtained for assays of RE and enzyme activity. The specific activity of lecithin:retinol acyltransferase (LRAT) was approximately 10-fold greater in the nonparenchymal cell than the hepatocyte fraction from both vitamin A-sufficient and retinoid-treated rats. Total RE mass, newly synthesized [3H]RE and LRAT activity were positively correlated in liver and isolated cells of both normal (P < 0.0001) and retinoid-treated rats (P < 0.0002). In nonparenchymal cells, these three constituents were nearly equally enriched as evaluated by their relative specific activity values (RSA, defined as the percentage of recovered activity divided by the percentage of recovered protein), which were each significantly greater than 1.0, with values of 4.3 for total RE mass (P < 0.05), 3.6 for newly synthesized [3H]RE (P < 0.01) and 3.8 for LRAT activity (P < 0.01). In contrast, the specific activities of neutral and acid bile salt-independent retinyl ester hydrolases (REH) did not vary with vitamin A status, and their RSA values were close to 1.0 in both hepatocytes and nonparenchymal cells. These data show that LRAT and REH are differentially regulated by retinoids and that these enzymes also differ in their spacial distribution between liver parenchymal and nonparenchymal cells.

Acyltransferases↗
PubMed1997

Molecular cloning of the cDNA for rat hepatic, bile salt-dependent cholesteryl ester/retinyl ester hydrolase demonstrates identity with pancreatic carboxylester lipase.

Rat liver homogenates contain a neutral lipid ester hydrolase that requires millimolar concentrations of bile salts for maximal activity in catalyzing the hydrolysis of cholesteryl esters and retinyl esters in vitro. Previous studies have demonstrated that this hepatic hydrolase resembles rat pancreatic carboxylester lipase because it reacts with a specific pancreatic carboxylester lipase antibody and the eight N-terminal amino acids of the hepatic protein are identical to those of the pancreatic enzyme. Nonetheless, the exact molecular relationship between the hepatic and pancreatic enzymes is unclear. In the present study, a rat hepatic cDNA encoding the enzyme was cloned. Sequence analysis demonstrated that this cDNA corresponds to the full-length mature pancreatic carboxylester lipase (EC# 3.1.1.13). In individual animals the hepatic and pancreatic cDNA sequences were identical. However, among rats there were sequence variations, suggesting a polymorphic nature for this rat gene.

Amino Acid Sequence↗
PubMed1995

Distributions of carotenoids and alpha-tocopherol among lipoproteins do not change when human plasma is incubated in vitro.

Carotenoids and alpha-tocopherol are dietary, lipophilic antioxidants which may protect plasma lipoproteins from oxidation, a process believed to contribute to atherogenesis. In this study, the quantities and distributions of carotenoids, alpha-tocopherol and major lipids in the plasma and lipoproteins of seven normolipidemic humans were determined. Experiments were also conducted to determine if these antioxidants redistribute among lipoproteins when plasma is incubated in vitro. Virtually all of the total carotenoid in plasma associated with lipoproteins, primarily LDL [73 +/- 10% (mean +/- SD)], as did the more non-polar individual carotenoids, beta-cryptoxanthin (68 +/- 9%); lycopene (79 +/- 9%), and beta-carotene (72 +/- 12%), in patterns which closely resembled the distribution of total cholesterol. Xanthophyll, the most polar carotenoid examined, distributed equally between LDL (44 +/- 11%) and HDL (38 +/- 14%), whereas alpha-tocopherol associated with LDL (43 +/- 12%), HDL (26 +/- 10%), and VLDL (27 +/- 13%). These patterns closely resembled that of phospholipid. Approximately four carotenoid molecules associated with each VLDL and one with each LDL particle, whereas only 25 of every 1000 HDL particles contained carotenoid. Approximately 145 molecules of alpha-tocopherol associated with VLDL, 12 with LDL, and one with each HDL particle. Unlike triglyceride and cholesteryl ester, known to transfer among lipoproteins through the action of cholesteryl ester transfer protein, net transfer of carotenoids and alpha-tocopherol among lipoproteins did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Hepatic uptake and metabolism of chylomicron retinyl esters: probable role of plasma membrane/endosomal retinyl ester hydrolases.

Previous studies have indicated the presence of both neutral and acid, bile salt-independent retinyl ester hydrolases associated with plasma membrane and endosome fractions of rat liver homogenates. In the present studies, chylomicrons containing tritium-labeled retinyl esters were injected intravenously into rats in order to study the initial metabolism of retinyl esters during and after uptake into the liver. At various times after chylomicron injection, plasma was obtained and the liver was homogenized and subjected to analytical subcellular fractionation. Labeled retinyl esters were rapidly cleared from plasma (half-time approximately 10 min) and appeared in the liver. Within the liver, label first appeared in plasma membrane/endosomal fractions that were also enriched in both neutral and acid, bile salt-independent retinyl ester hydrolase activities. At no time were the labeled esters significantly associated with fractions enriched in lysosomes. Rather, it appeared that the labeled esters were hydrolyzed and/or transferred to fractions enriched in endoplasmic reticulum. These studies demonstrate the co-localization of newly delivered retinyl esters and bile salt-independent retinyl ester hydrolase enzyme activities and thus, suggest a probable role for these enzymes in the initial hepatic metabolism of chylomicron retinyl esters. This conclusion was further supported by the observation that plasma membrane/endosomal fractions were active in catalyzing the hydrolysis of chylomicron remnant retinyl esters in vitro.

Animals↗
PubMed1994

Bile salt-dependent and bile salt-independent cholesteryl ester hydrolase activities in rat liver cytosol.

These studies report on the relationship between the bile salt-dependent and -independent cholesteryl ester hydrolase (CEH) activities found in rat liver cytosol. The two activities show very similar Michaelis-Menten substrate kinetics and pH dependence. After gel filtration of cytosol, the bile salt-independent activity elutes much earlier than the bile salt-dependent activity, suggesting that the two activities are associated with entities of different molecular size. However, when gel filtration is carried out in the presence of bile salt, the bile salt-dependent activity elutes as a large aggregate, similar to the bile salt-independent activity's behavior in the absence of bile salt. Both activities coelute after cytosol is passed through an ion exchange column. After each chromatographic procedure the recovery of the bile salt-dependent activity was substantially higher than the recovery of the bile salt-independent activity. When cytosol is incubated with anti-rat pancreatic CEH in the absence of cholate, the bile salt-dependent activity is inhibited more than 90% whereas bile salt-independent activity remains unaffected even at high antibody concentrations. When cytosol is incubated with anti-rat pancreatic CEH in the presence of cholate both CEH activities remain unaffected. The prevention of immunoinhibition by cholate seems to be specific for this detergent since CHAPS, a cholate analog, does not prevent immunoinhibition of the bile salt-dependent activity by anti-CEH. The experimental results are consistent with a model for CEH activity in liver cytosol in which there is only one enzyme that can exist in a monomeric, inactive form (that can be activated by addition of cholate to the assay and represents the bile salt-dependent activity) and in an active complex comprising several enzyme monomers as well as cholate micelles (that accounts for the bile salt-independent activity).

Animals↗
PubMed1993

Analysis of microsomal cholesteryl ester hydrolases by radiation inactivation.

Radiation inactivation by high energy electrons, a method for determining the size of a protein without prior purification, was used to study the acid and neutral cholesteryl ester hydrolase (CEH) activities of rat liver microsomes. The same preparations were also assayed for the microsomal, "nonspecific" carboxylesterases using o-nitrophenyl acetate as substrate. Non-specific esterase activity surviving radiation could be fit to a single exponential function, the slope of which yielded a target size of 47 +/- 5 kDa (mean +/- S.D., n = 7). Surviving CEH activity assayed at pH 5 could also be fit to a single exponential that yielded a target size of 71 +/- 14 kDa (n = 5). In contrast, the surviving CEH activity assayed at pH 7 was more complex. The data from six experiments were described as the sum of two exponentials, indicating that most of the activity is due to an entity that is three to four times larger and a minor amount to one that is half the size of the pH 5 enzyme. The results are consistent with the suggestion that the acid and neutral microsomal CEH activities are due to distinct enzymes, which are not the "nonspecific" carboxylesterases. Their sizes also differ from those previously determined for lysosomal acid lipase and other lipases in the liver.

Animals↗
PubMed1992

Characterization of a bile salt-dependent cholesteryl ester hydrolase activity secreted from HepG2 cells.

HepG2 cells and medium were assayed for cholesteryl ester hydrolase (CEH) activity in the presence and absence of sodium cholate. Although bile salt-dependent CEH activity was measured in the medium at 6 to 96 h (up to 4500 pmol/h per mg cell protein), there was very little activity detected in the corresponding cell homogenates (less than 70 pmol/h per mg cell protein). Activity in the medium was expressed only in the presence of trihydroxy bile salts and was maximal at 40 mM cholate and pH 7.5. Incubation of HepG2 cells with brefeldin A resulted in an 80 to 90% inhibition of secretion of the bile salt-dependent CEH activity, while only inhibiting total protein secretion by 42%. Bile salt-dependent CEH activity could also be detected in rat liver perfusates. Although there was measurable activity in all of 14 livers analyzed (47 +/- 10 and 53 +/- 17 nmol/h per g liver per h perfusion during two 5-min collections after 15 and 30 min of perfusion, respectively), it did not correlate with the activity found in corresponding liver homogenates, as only four livers had detectable bile salt-dependent CEH activity. These results provide evidence for the secretion of a bile salt-dependent CEH activity, from both a hepatic cell line and the intact liver, that has similar properties to the enzyme previously isolated from rat liver homogenates and rat pancreas.

Animals↗
PubMed1992

Tissue and species differences in bile salt-dependent neutral cholesteryl ester hydrolase activity and gene expression.

Enzymatic activity and mRNA abundance for neutral bile salt-dependent cholesteryl ester hydrolase (CEH) were determined in rat and rabbit tissues. In rat liver and intestine, enzyme activity and mRNA levels varied independently. Particularly striking in most tissue samples was the absence of detectable CEH mRNA in the presence of enzymatic activity, suggesting that there was an exogenous source of enzyme. Rabbits differed from rats in four ways. First, neither CEH activity nor mRNA was present in any liver sample. Second, CEH mRNA was present in nearly all intestinal samples, and its abundance tended to correlate with enzymatic activity. Third, rabbit CEH mRNA was approximately 250 bases shorter than the rat message. Fourth, we have previously shown that rat plasma contains CEH activity, whereas in the present studies, rabbit plasma did not contain such activity. Overall, our studies indicate that CEH activity in rat liver, intestine, and plasma can be derived exogenously, most likely from the uptake and transport of pancreatic enzyme. In contrast, in rabbit the lack of CEH activity in plasma and liver and the capacity of the intestine for in situ synthesis of CEH suggest that this animal does not have the same ability to distribute pancreatic CEH. These species differences in CEH metabolism may partly explain the greater susceptibility of rabbit tissues to accumulate cholesteryl esters.

Animals↗
PubMed1991

Differential effects of fish oil, safflower oil and palm oil on fatty acid oxidation and glycerolipid synthesis in rat liver.

Studies were conducted to explore the mechanisms by which dietary fish oil decreases hepatic triglyceride secretion. Forty-five rats (15/group) were fed purified diets containing 10% fat as either fish oil, safflower oil or palm oil for 10 d. Plasma triglyceride concentration was lowest in the fish oil-fed group followed by the groups fed safflower oil and palm oil. The liver's capacity to oxidize fatty acids was assessed by assays of mitochondrial and peroxisomal beta-oxidation pathways in whole homogenates. Additionally, key enzymatic activities in the biosynthesis of triglyceride (diacylglycerol acyltransferase, phosphatidate hydrolysis) and phosphatidylcholine (CTP:phosphocholine cytidylyltransferase) were assayed. Compared with those fed palm oil the fish oil-fed animals showed 25% greater mitochondrial beta-oxidation but this difference was not statistically significant (P = 0.1). Fish oil feeding led to 45% greater (P less than 0.05) peroxisomal beta-oxidation. Diacylglycerol acyltransferase activity was unaffected by the type of dietary fat and slightly (13%) but significantly (P less than 0.02) lower cytidylyltransferase activity due to fish oil feeding was observed. More strikingly, both fish oil and safflower oil diets significantly lowered phosphatidate hydrolysis by 37 and 22%, respectively, compared with the palm oil diet. This activity directly correlated (r = 0.68; P less than 0.001) with plasma triglyceride concentration. Thus, dietary fish oil might suppress triglyceride secretion by decreasing glycerolipid synthesis, an effect mediated by changes in one or more enzymes involved in phosphatidate catabolism.

Animals↗
PubMed1991

Neutral and acid retinyl ester hydrolases associated with rat liver microsomes: relationships to microsomal cholesteryl ester hydrolases.

We recently reported the presence of a neutral, bile salt-independent retinyl ester hydrolase (REH) activity in rat liver microsomes and showed that it was distinct from the previously studied bile salt-dependent REH and from nonspecific carboxylesterases (Harrison, E. H., and M. Z. Gad. 1989. J. Biol. Chem. 264: 17142-17147). We have now further characterized the hydrolysis of retinyl esters by liver microsomes and have compared the observed activities with those catalyzing the hydrolysis of cholesteryl esters. Microsomes and microsomal subfractions enriched in plasma membranes and endosomes catalyze the hydrolysis of retinyl esters at both neutral and acid pH. The acid and neutral REH enzyme activities can be distinguished from one another on the basis of selective inhibition by metal ions and by irreversible, active site-directed serine esterase inhibitors. The same preparations also catalyze the hydrolysis of cholesteryl esters at both acid and neutral pH. However, the enzyme(s) responsible for the neutral REH activity can be clearly responsible for the neutral REH activity can be clearly differentiated from the neutral cholesteryl ester hydrolase(s) on the basis of differential stability, sensitivity to proteolysis, and sensitivity to active site-directed reagents. These results suggest that the neutral, bile salt-independent REH is relatively specific for the hydrolysis of retinyl esters and thus may play an important physiological role in hepatic vitamin A metabolism. In contrast to the neutral hydrolases, the activities responsible for hydrolysis of retinyl esters and cholesterol esters at acid pH are similar in their responses to the treatments mentioned above. Thus, a single microsomal acid hydrolase may catalyze the hydrolysis of both types of ester.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Synthesis and degradation of fatty acid ethyl esters by cultured hepatoma cells exposed to ethanol.

Fatty acid ethyl esters are a family of neutral lipids that are the products of esterification of fatty acids with ethanol. Unlike other pathways of ethanol metabolism, ethyl esters are present in numerous human organs which are the targets of ethanol-induced damage. In the present study, we have shown that fatty acid ethyl esters are synthesized by a hepatoma cell line in tissue culture when exposed to ethanol concentrations easily attained by man during social drinking. Unlike alcohol dehydrogenase, the enzyme(s) responsible for synthesis of ethyl esters are membrane-bound and concentrated in the microsomal fraction of rat hepatocytes. In addition, fatty acid ethyl esters are hydrolyzed to free fatty acids and ethanol by membrane-bound enzyme(s) that are enriched in the microsomal and mitochondrial-lysosomal fractions. Intracellular hydrolysis of fatty acid ethyl esters release free fatty acids which are preferentially incorporated into cellular cholesterol esters. Thus, we have shown that a hepatocellular line exposed to concentrations of ethanol easily achieved in man by social drinking utilize endogenous fatty acids to form long-lived ethanol metabolites, fatty acid ethyl esters. Importantly, this family of neutral lipids may act as biochemical mediators of ethanol-induced cell damage, including the changes in cholesterol metabolism noted in chronic alcoholics.

Animals↗
PubMed1990

Inhibitors of neutral cholesteryl ester hydrolase.

p-Nitrophenyl N-butyl, N-octyl, and N-dodecyl carbamates and a newly synthesized diethyl phosphate compound were studied as potential inhibitors of the cholesteryl ester hydrolases of Fu5AH rat hepatoma cells. Whole homogenates of Fu5AH cells were used as an enzyme source for the assay of cholesteryl ester hydrolase activity. All four compounds led to marked inhibition (70-80%) of neutral cholesteryl ester hydrolase activity (assayed at pH 7) at concentrations where the activity of acid cholesteryl ester hydrolase (assayed at pH 4) was unaffected. Cholesteryl ester hydrolysis was also evaluated in intact cultured cells induced to accumulate cholesteryl esters in cytoplasmic lipid droplets by exposure to cholesterol-rich phospholipid dispersions. Hydrolysis was then assessed during subsequent incubations in the presence of an inhibitor of cholesterol esterification. All compounds caused significant inhibition of cholesterol ester hydrolysis with the diethyl phosphate being the most effective. At a concentration that caused greater than 90% inhibition of the hydrolysis of cytoplasmic cholesteryl esters, the compound had only a minimal effect on lysosomal hydrolysis of cholesteryl esters. These results suggest that diethyl phosphates and N-alkylcarbamates may be of value in future studies on the substrate specificities, regulation, and physiological role(s) of cholesteryl ester hydrolases.

Animals↗
PubMed1989

Hydrolysis of retinyl palmitate by enzymes of rat pancreas and liver. Differentiation of bile salt-dependent and bile salt-independent, neutral retinyl ester hydrolases in rat liver.

Previous studies have demonstrated that homogenates of the livers of rats contain a neutral retinyl ester hydrolase activity that requires millimolar concentrations of bile salts for maximal in vitro activity. The enzymatic properties of this neutral, bile salt-dependent retinyl ester hydrolase activity in liver homogenates are nearly identical to those observed in the present report for the in vitro hydrolysis of retinyl palmitate by purified rat pancreatic cholesteryl ester hydrolase (EC 3.1.1.13). Moreover, anti-rat pancreatic cholesteryl ester hydrolase IgG completely inhibits the bile salt-dependent retinyl ester hydrolase activity of rat liver homogenates whereas normal rabbit IgG does not. We also show that liver homogenates contain a neutral, bile salt-independent retinyl ester hydrolase activity that differs from the bile salt-dependent activity in that 1) its absolute activity does not vary markedly among individual rats, 2) it is not inhibited by antibodies to pancreatic cholesteryl ester hydrolase, and 3) it is localized in the microsomal fraction of liver homogenates. Subfractionation of microsomes demonstrates that the neutral, bile salt-independent retinyl ester hydrolase activity is associated with liver cell plasma membranes and thus may play a role in the hydrolysis of retinyl esters delivered to the liver by chylomicron remnants.

Animals↗
PubMed1989

Expression in Xenopus oocytes of rat liver mRNA coding for a bile salt-dependent cholesteryl ester hydrolase.

A catalytically active bile salt-dependent cholesteryl ester hydrolase (CEH) was expressed when Xenopus oocytes were injected with rat liver mRNA. The expressed CEH activity was highly dependent on the presence of trihydroxy bile salts (cholate or one of its conjugates); maximum hydrolytic activity was observed in the presence of 10 mM sodium cholate. The expressed CEH was not activated by dihydroxy bile salts (deoxycholate and its conjugates). In the presence of 10 mM sodium cholate, the CEH activity was maximal near pH 7 but was significant between pH 6 and 8. Monospecific immune IgG raised against rat pancreatic CEH completely inhibited the CEH expressed in Xenopus oocytes. Phenylmethylsulfonyl fluoride, a serine enzyme inhibitor, was inhibitory to the expressed CEH activity, whereas p-chloromercuribenzoate (up to 5 mM), a potent thiol-blocking agent, did not significantly inhibit the expressed activity. These experiments clearly demonstrate that the liver contains an mRNA encoding a bile salt-dependent CEH activity and suggest that the uptake of pancreatic enzyme is not necessarily the source of liver CEH as has been speculated.

Animals↗
PubMed1988

Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein.

We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls.

Animals↗
PubMed1988

Bile salt-dependent, neutral cholesteryl ester hydrolase of rat liver: possible relationship with pancreatic cholesteryl ester hydrolase.

Homogenates of the livers of outbred, Sprague-Dawley rats contain a neutral cholesteryl ester hydrolase activity that requires millimolar concentrations of bile salts for maximal activity. Previous studies showed that this activity had the unusual property of being highly variable among individual rats. The present studies were conducted to define further the nature of this enzymic activity and to explore the possible basis for the variability. Individual liver homogenates from inbred Fisher-344 rats showed the same range and magnitude of activity as outbred rats, suggesting that genetic heterogeneity was not a factor in determining the enzyme activity. Tissue distribution studies showed the presence of a very similar enzyme activity in serum, bile and intestinal homogenates, with the specific activity in intestine being 25-500-times greater than that in liver. Moreover, the enzymic properties of the activity in serum, liver and intestine were identical to those of purified rat pancreatic cholesteryl ester hydrolase (EC 3.1.1.13). Monospecific, anti-pancreatic hydrolase IgG specifically and completely inhibited the cholesteryl ester hydrolase activity in rat serum, intestine and liver. The results raise the possibility that the neutral, bile salt-dependent cholesteryl ester hydrolase activity of rat liver homogenates may be due to the uptake of enzyme originating in the pancreas. This, in turn, may explain the dramatic variation in activity observed among individual rat livers.

Animals↗